Engineered nucleases, including zinc finger nucleases, TALENs, CRISPR/Cas nuclease systems, and homing endonucleases designed to specifically bind to target DNA sites are useful in genome engineering. For example, zinc finger nucleases (ZFNs) and TALENs (including TALENs comprising Fok1-TALE DNA binding domain fusions, Mega TALs and compact TALENs) are proteins comprising engineered site-specific zinc fingers or TAL-effector domains fused to a nuclease domain ZFNs and TALENs have been successfully used for genome modification in a variety of different species. See, for example, U.S. Pat. Nos. 7,888,121; 8,409,861; 8,586,526; 7,951,925; 8,110,379; 7,919,313; 8,597,912; 8,153,399; 8,399,218; and United States Patent Publications 20090203140; 20100291048; 20100218264; and 20110041195, the disclosures of which are incorporated by reference in their entireties for all purposes. Additionally, the CRISPR/Cas system can be manipulated through use of an engineered crRNA/tracr RNA (‘single guide RNA’) to perform genome engineering (Jinek et al. (2012) Science 337 p 816-821). See, for example, U.S. Publication No. 20150056705.
These engineered nucleases and engineered nuclease systems can create a double strand break (DSB) in a target nucleotide sequence, which increases the frequency of homologous recombination at the targeted locus by more than 1000-fold. In addition, the inaccurate repair of a site-specific DSB by non-homologous end joining (NHEJ) and other pathways can also result in gene disruption.
In mammalian and plant cells these DNA lesions are repaired by an extensive array of well-characterized DNA-repair pathways. See, e.g., Ciccia and Elledge (2010) Mol. Cell. 40(2):179-204 and Puchta (2005) J Exp Bio 56(409): 1-14. The choice of these pathways depends both on the DNA lesion type and on the status of the cell cycle with a preference for non-homologous end joining (NHEJ) in G1 phase and homology-directed repair (HDR) during or after S-phase. But even for a defined lesion in a defined cell cycle status, the cell can choose from a variety of molecular tools for repair. These pathways are thought to follow a hierarchy which first prefers error-free pathways and secondary, as a last resort, error-prone pathways.
For the use of nucleases such as ZFNs and TALENs or nuclease systems such as CRIPSR/Cas, in gene therapy or genome engineering, the desired repair outcome at the site of the cleavage is either gene disruption (e.g., inactivation) or gene correction. See, e.g., Urnov et al. (2010) Nat Rev Genet. 11(9):636-46. The vast majority of the 5′ four base overhangs that are generated by artificial nucleases comprising Fok1 cleavage domains in vivo and in vitro are repaired error-free by classic DNA-PKcs dependent NHEJ (also termed “C-NHEJ”) rather than by the more error-prone alternative NHEJ (“A-NHEJ”). It has been shown that A-NHEJ can be carried out by a complex including Poly-(ADP-ribose) polymerase 1 (PARP1), an enzyme which also contributes to single strand break (SSB) repair. Alternatively, DSBs can also be repaired by other, even more error-prone pathways like microhomology-mediated end joining (MMEJ) which is known to use small DNA sequence homologies and DNA end-resection at the site of damage (shown in FIG. 1). DSB repair pathways follow a hierarchy of activation from error-free to error-prone repair, so in order to achieve error-prone repair, the error-free pathway must first be inhibited.
Mammalian and plant cells can also use HDR if a DNA repair template is available. This repair template can either be a homologous chromosome, a sister chromatid or, in the case of gene therapy, a transfected single or double-stranded DNA donor template with any gene sequence (e.g., transgene) as long as the donor contains regions of homology with the targeted sequence. In order to achieve gene correction via HDR the cells must either be in S-phase where HDR is preferred over NHEJ or the cell must exhaust all its NHEJ-like repair options before resorting to HDR. Another possible scenario for HDR induction is the persistence of DNA damage inflicted during G1 until S-phase. If there are persistent SSBs and DSBs that are encountered by DNA replication forks during DNA replication, the replication forks can collapse, form DSBs which, subsequently, are repaired by HDR directed repair.
Thus, there remains a need for methods and compositions that shift nuclease-mediated error-free DNA repair to both error-prone and HDR-mediated DNA repair events to enhance nuclease-mediated gene disruption and targeted integration.